Abstract

BACKGROUND:

Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs). Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs. Substrate reduction therapy is one of potential treatment of these diseases. It was demonstrated previously that genistein (4', 5, 7-trihydroxyisoflavone) inhibits synthesis and reduces levels of GAGs in cultures of fibroblasts of MPS patients. Recent pilot clinical study indicated that such a therapy may be effective in MPS III (Sanfilippo syndrome).

METHODS:

To learn on details of the molecular mechanism of genistein-mediated inhibition of GAG synthesis, efficiency of this process was studied by measuring of incorporation of labeled sulfate, storage of GAGs in lysosomes was estimated by using electron microscopic techniques, and efficiency of phosphorylation of epidermal growth factor (EGF) receptor was determined by using an ELISA-based assay with fluorogenic substrates.

RESULTS:

Effects of genistein on inhibition of GAG synthesis and accumulation in fibroblasts from patients suffering from various MPS types were abolished in the presence of an excess of EGF, and were partially reversed by an increased concentration of genistein. No such effects were observed when an excess of 17beta-estradiol was used instead of EGF. Moreover, EGF-mediated stimulation of phsophorylation of the EGF receptor was impaired in the presence of genistein in both wild-type and MPS fibroblasts.

CONCLUSION:

The results presented in this report indicate that the mechanism of genistein-mediated inhibition of GAG synthesis operates through epidermal growth factor (EGF)-dependent pathway.

Effects of genistein, EGF, 17β-estradiol and combinations of genistein with EGF or 17β-estradiol on GAG synthesis in MPS IIIA fibroblasts. Cells were untreated (Ctrl) or treated with genistein at concentrations 10 or 30 μM (G10 and G30, respectively) alone, EGF alone at 100 ng/ml (EGF), 17β-estradiol alone at 1 nM (Est) or in following combinations: genistein (10 or 30 μM) with EGF (100 ng/ml) (EGF+G10 and EGF+G30, respectively) and genistein (10 or 30 μM) with 17β-estradiol (1 nM) (Est+G10 and Est+G30, respectively). Cell cultures were labeled with 20 μCi/ml of [35S]Na2SO4 for 24 h, and GAG synthesis was monitored by measuring incorporation of 35S into proteoglycans. The results were calculated per DNA amount. The results are mean values of three experiments with error bars indicating SD.

Short-term effects of EGF and genistein on phosphorylation of the EGF receptor. Fibroblasts were cultured in the absence of EGF, which indicated the residual phosphorylation level (baseline value), and in the presence of EGF (100 ng/ml for 5 min) without any inhibitor (Ctrl) or with genistein (either 10 or 30 μM, marked as G10 or G30, respectively) or PD168393 (either 10 or 30 nM, marked as Inh10 or Inh30, respectively) added 30 min before EGF. Results of experiments with HDFa (wild-type), MPS II and MPS IIIB cell (fibroblast) lines are shown. Other tested fibroblasts gave similar results (results not shown). The results were calculated as phospho-EGF receptor fluorescence at 600 nm normalized to the total EGF receptor fluorescence at 450 nm. Therefore, the obtained normalized values are in arbitrary units. Values represent mean ± range of duplicate determinations.

Long-term effects of EGF and genistein on phosphorylation of the EGF receptor. Fibroblasts were cultured in the absence of EGF, which indicated the residual phosphorylation level (baseline value), and in the presence of EGF (100 ng/ml for 24 h) (Ctrl) without genistein or with genistein (either 10 or 30 μM, marked as G10 or G30, respectively) added together with EGF. Results of experiments with HDFa fibroblasts (wild-type) are shown. Similar effects were observed in experiments with MPS II, MPS IIIA and MPS IIIB fibroblasts (results not shown). The results were calculated as phospho-EGF receptor fluorescence at 600 nm normalized to the total EGF receptor fluorescence at 450 nm. Therefore, the obtained normalized values are in arbitrary units. Values represent mean ± range of duplicate determinations.

Effects of the length of incubation of cell cultures with genistein on inhibition of EFG receptor phosphorylation. Experiments were performed as described in Methods and in captions to Figs. 3 and 4. Genistein was used at final concentrations of 10 μM (black columns) or 30 μM (grey columns). Results of experiments with HDFa (panel A) and MPS II (panel B) fibroblasts are shown. Other tested fibroblasts gave similar results (results not shown). Values represent mean ± range of duplicate determinations.